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Geometric and Histopathologic Assessment of Yarrow Extracts (Achillea millefolium) and on Healing of Experimental Skin Wounds and its Comparison with Zinc Oxide on Rats

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Skin wound healing especially when it occurs on the face or somewhere important for cosmetic reasons, is very significant physiological procedure. It is quite obvious that promoting this healing is important too. In this study we tried to compare the effects of Achillea millefolium and Zinc oxide on secondary intentioned open-wound healing in rats. Zinc oxide is being used worldwide as an absorbent and protective compound. Its pharmacological properties are wide and its non-toxic material allows it to be used as a routine skin care substance. In current study, 70 female wistar rats where included in 5 groups. Full thickness Incisional wound with 23 mm diameter was made with surgical scissors and scalpel. The whole operation was taking place under general Anesthesia and analgesia circumstances. After making surgical wounds, rats are treated as mentioned in the text. Rats are observed for 28 days for wound closure process and inflammatory conditions taking place in wound. Biopsy intervals are 0 (the day of surgery), 3, 7, 14, 21 and 28th day after surgery. In these certain days rats were euthanized and biopsies of wound sites were obtained. Wounds areas are also measured by Scion Image ™ software daily. At last, all data were analyzed using SPSS statistics ver.17. As a result, Achillea millefolium at the dose of 10% has significant healing properties compared to Zinc oxide. These data were validating under confidence surface of 95% (p<0.01).
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Current Research Journal of Biological Sciences 4(1): 90-95, 2012
ISSN: 2041-0778
© Maxwell Scientific Organization, 2012
Submitted: November 18, 2011 Accepted: December 20, 2011 Published: January 20, 2012
Corresponding Author: Ali Rezaie, Department of Clinical Science, Tabriz Branch, Islamic Azad University, Tabriz, Iran
90
Geometric and Histopathologic Assessment of Yarrow Extracts
(Achillea millefolium) and on Healing of Experimental Skin Wounds and its
Comparison with Zinc Oxide on Rats
1Ali Rezaie, 2Daryoush Mohajeri, 3Mohammadreza Valilou, 1Ghafour Mousavi, 4Mehrdad Nazeri,
5Babak Mohammadi and 5Solmaz Zakhireh
1Department of Clinical Science, Tabriz Branch, Islamic Azad University, Tabriz, Iran
2Department of Pathobiology, Tabriz Branch, Islamic Azad University, Tabriz, Iran
3Department of Veterinary Medicine, Shabestar Branch, Islamic Azad University, Shabestar, Iran
4Young Researchers Club, Tabriz Branch, Islamic Azad University, Tabriz, Iran
5Department of Basic Science, Faculty of Chemistry, Ahar Branch, Islamic Azad University,
Ahar, Iran
Abstract: Skin wound healing especially when it occurs on the face or somewhere important for cosmetic
reasons, is very significant physiological procedure. It is quite obvious that promoting this healing is important
too. In this study we tried to compare the effects of Achillea millefolium and Zinc oxide on secondary
intentioned open-wound healing in rats. Zinc oxide is being used worldwide as an absorbent and protective
compound. Its pharmacological properties are wide and its non-toxic material allows it to be used as a routine
skin care substance. In current study, 70 female wistar rats where included in 5 groups. Full thickness Incisional
wound with 23 mm diameter was made with surgical scissors and scalpel. The whole operation was taking
place under general Anesthesia and analgesia circumstances. After making surgical wounds, rats are treated
as mentioned in the text. Rats are observed for 28 days for wound closure process and inflammatory conditions
taking place in wound. Biopsy intervals are 0 (the day of surgery), 3, 7, 14, 21 and 28th day after surgery. In
these certain days rats were euthanized and biopsies of wound sites were obtained. Wounds areas are also
measured by Scion Image ™ software daily. At last, all data were analyzed using SPSS statistics ver.17. As
a result, Achillea millefolium at the dose of 10% has significant healing properties compared to Zinc oxide.
These data were validating under confidence surface of 95% (p<0.01).
Key words: Achillea millefolium, healing, rats, skin wounds, zinc oxide
INTRODUCTION
The genus Achillea (Asteraceae), named after the
mythological Greek warrior Achilles, who used Achillea
species for healing wounded-soldiers during the Trojan
War (Cheers, 1999). The genus Achillea comprises of -85
species, most of which are endemic to Europe and the
Middle East. Turkish flora possesses 42 Achillea species
and 23 of them are endemic (Duman and Achillea, 2000).
These species have some interesting properties and are
used in cosmetics, fragrances and agriculture, for
example, plant protection (Senatore et al., 2005). Some
Achillea species have been known to be
ethnopharmacologically used in folk remedies for various
purposes such as hemorrhoid and wound healing (Baytop,
1999). Herbal teas prepared from some Achillea species
are very often used in folk medicine as diuretic, for
abdominal pain, against diarrhea, flatulence and
emmenagog, moreover for wound healing purposes
(Fujita et al., 1995; Honda et al., 1996; Yesilada et al.,
1993). Achillea biebersteinii is locally named yarrow, and
other species widely used as a folk remedy to treat
abdominal pain, wounds and stomachache as well (Sezik
et al., 2001; Baytop, 1997). A. biebersteinii Afan.
[Asteraceae, Section: Filipendulinae (D.C.) Boiss] (syn.
A. micrantha) is a perennial herb, villose, stems erect,
simple or branched from the base; 30-60 cm high; leaves
up to 10 cm, oblong-lanceolate in outline, pinnatisect into
numerous narrow segments, segments divided into minute
linear-lanceolate mucronate lobes; the heads are radiate,
in large dense compound corymbs; involucre 4-5 mm,
oblong-ovoid; flowering period, April-May. Several
biological activity studies have been performed on various
Achillea species, including antibacterial, antioxidant, anti-
inflammatory and antispasmodic activities (Karamenderes
and Apayd2n, 2003; Candan et al., 2003; Al-Hindawi
et al., 1989; Skocibusic et al., 2004).
Zinc oxide is an inorganic compound with the
formula ZnO. It is a white powder that is insoluble in
water. Zinc is an essential trace element of which about 2
g is found in the adult human body. At least 200 enzymes
in different biological systems are dependent on the
presence of the zinc ion. Among these zinc-dependent
enzymes, DNA and RNA polymerases are crucial during
Curr. Res. J. Biol. Sci., 4(1): 90-95, 2012
91
tissue repair as they affect cell proliferation and protein
synthesis. In accordance with the biochemical role of zinc
a reduced synthesis of DNA, reduced deposition of
granulation tissue, decreased tensile strengths in skin
incisions, and delayed closure rates in excised wounds
in zinc-deficient rats have been demonstrated
(Sandstead et al., 1970; Prasad and Oberleas, 1974). Zinc
supplementation restored to normal the tensile Strengths
of the incisional and healing rates of the excisional
wounds (Sandstead et al., 1970). It has been clinically
shown that the healing of leg ulcers is delayed in patients
with subnormal serum-zinc levels (Haley, 1979). Zinc
given as oral and topical zinc sulfate or as topical zinc
oxide normalizes impaired healing ability in these patients
(Haley, 1979; Golden et al., 1980; Stromberg and Agren,
1984).
The aim of the present study was to investigate the in
vivo wound healing activity of A. millefolium in order to
elucidate traditional use of this plant from the scientific
point of view.
MATERIALS AND METHODS
Animals: This study was conducted in Islamic Azad
university research center during summer 2011. In this
study, 70 male wistar rats weighted 210±10 g and aged 12
weeks old were selected. All animals were kept in same
situation (temperature 24ºC and humanity 70%) and food
and water were provided ad libitum.
Pre-operation measures: The operation (induction
wound in the skin) required general anesthesia, analgesia
and muscle relaxation. In term, we used of Ketamine
(10%, 60 mg/kg) and xylazine (2%, 10 mg/kg) through
IM injection to induction of anesthesia and pre-operation
drugs, respectively. To prevention of drugs side effects,
liquid therapy with dextrose 5% at the dose of 50-100
mg/kg/day was exerted immediately after induction of
anesthesia.
Operation measures: After preparation the dorsal skin of
rats (distinct between scapula to ischial tuberosity), a
wound in circle shaped with 7 mm in diameter and by
biopsy punch were inducted. In this study rout of
wounding was excisional wounding that in way
epidermis, dermis, hypoderm and Panniculus Carnosus
completely were removed. After wounding, rats were
divided into 5 groups of 15.
Group 1: received high doses (20%) of herbal extract
Group 2: received low doses (10%) of herbal extract
Group 3:as positive control group received zinc oxide
20%
Group 4: as negative control group received eucerin
Group 5: as control not received any drug
Samples were fixed in the formalin 10% and sent to
pathology laboratory.
Post-operation measures: After biopsy and washing
wound area with normal saline, all drugs were
administrated as local way by an applicator in the wound
area. This administration continued for 21 days.
Sampling: On days 0, 3, 7, 14, 21 and 28 of research,
samples as tissue specimens from biopsy areas were
collected and sent to pathology laboratory. Sampling was
done under anesthesia condition and this anesthesia was
induced by Ketamine and Rampon. Sampling was exerted
by scalpel. Samples were fixed into formalin 10%. In lab,
after processing and staining to H&E method slides were
achieved. Slides were investigated by a light microscope.
Statistical analysis: The Statistical Package for Social
Sciences (SPSS Inc., Chicago, IL, USA), version 17.0,
was used for statistical analysis. All data are presented as
mean ±SEM. Before statistical analysis, all variables were
checked for normality and homogeneity of variance by
using the Kolmogorov-Smirnoff and Levene tests,
respectively. The data obtained were tested by ANOVA
followed by Tukey's post-hoc multiple comparison test.
p<0.05 was considered statistically significant.
RESULTS
Geometric findings: On the first day of trial, ulcer size in
all five groups showed a significant increase compared to
day zero. On the second day, wound size in high dose
treatment group was reduced significantly. So that this
finding was observed in low dose and zinc oxide groups,
control group and eucerin group on days 3, 4 and 5
respectively. Over seven days until day 7, according to the
size of the wound, the highest rate of wound shrinkage
was observed in high dose, low dose, zinc oxide, eucerin
and control group respectively. On day 21, maximum and
minimum shrinkage was observed in low dose and control
groups, respectively.
Histopathologic findings: On day 3 in high dose
treatment group, pustule covered the wound but still
retains its moisture. Re-epithelialization is seen from
wound sides. Inflammatory cells also are existed.
Infiltration of fibroblasts into the connective tissue was
obvious. In low dose treatment group, wound was covered
by pustule consist of fibrin and blood cells and purulent
materials such as neutrophils and RBC remnants. Clod on
the wound had more and low inflammatory cells than high
dose and zinc oxide groups, respectively. In zinc oxide
group, wound was covered by thick and keratinous
pustule. Wound was filled with granular connective tissue
and hyperemia was obvious. In eucerin group,
hemorrhage in the profound layers was obvious and was
not seen any pustule and healing has not been started
(Fig. 1).
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92
Fig. 1: (A) Microscopic view from wound area in healing from high dose group on day 3. Arrow shows Re-epithelialization from
wound sides, (B) Microscopic view from wound area in healing from high dose group on day 3. H&E 100x
Fig. 2: (A) Microscopic view from wound area in healing from high dose group on day 7. Arrows shows Re-epithelialization from
wound sides. H&E 40x, (B) Microscopic view from wound area in healing from group 4 on day 7. Arrows shows expanding
of epithelial cells into the pustule. H&E 100x
Fig. 3: (A) microscopic view from wound area in healing from group 2 on day 14. H&E 80x, (B) microscopic view from wound
area in healing from group 3 on day 14. H&E 80x
On day 7, in high dose treatment group, in some
cases, pustules on the wound still have not been
completely dried but wound area has been filled with
multicellular and vascular granular tissue. Epithelial
regeneration continues and the amount of inflammatory
cells is also greatly reduced. In low dose treatment group,
wound surface covered by pustules and internal space of
wound in the middle parts filled by fibrinous and granular
connective tissue and inflammatory and purulent cells are
seen between fibrin and connective tissue and granular
Curr. Res. J. Biol. Sci., 4(1): 90-95, 2012
93
Fig. 4: (A) microscopic view from wound area in healing from group 2 on day 21. H&E 220x, (B) microscopic view from wound
area in healing from group 3 on day 21. Arrows shows skin appendix H&E 60x
texture is full of newly built vessels. In zinc oxide group,
situation is entirely like with low dose treatment group. In
eucerin treatment group, wound surface is covered by
pustules and regenerative epithelial cells starts to
expanding on to the wound surface from sides.
Inflammatory cells are purulent and infiltration of
fibroblasts and existence of newly built vessels indicates
formation of new granular tissue. In control group,
granular tissue as vascular and low filament tissue are
seen. Hemorrhage and hyperemia is also seen in Fig. 2.
On day 14, in high dose treatment group granular
tissue is existed in the wound area and newly formed
vessels are low than previous days. The intensity of
inflammatory cells is reduced. Collagen fibers are more
delicate and have more organization. Hydropic
degeneration is also seen in some of the epithelial cells. In
low dose treatment group, more thickly epithelial covered
wound surface. Collagen is thicker and has more
organization. Coagulum isn’t existed. In zinc oxide group,
fibroblasts start to synthesis of collagen. Inflammatory
cells reduced and newly formed vessels increased. Lining
tissue is seen in margin of the wound but clot is seen in
some places. Blood clot on the wound contains large
amounts of acute inflammatory cells were neutrophils and
RBC has penetrated into the clot. In eucerin group, space
of wound around is occupied by young and multicellular
tissue and regeneration of the lining tissue starts from
sides. Wound surface covered by pustules that follows
contains hyperemia granular tissue. In control group,
Marginal parts of the wound are completely covered by
epithelial tissue. Also, new and hyper cellular connective
tissue covered dermal layer (Fig. 3).
On day 21, in high dose treatment group, in some
cases wound surface is covered by lining tissue but in
some others this is not occurred completely. The severity
of inflammation and hydropic degeneration is reduced. In
low dose treatment group, partial edema and hyperemia is
still seen and collagen fibers were thicker and condensed
and were more organized than day 14. In zinc oxide
group, dermal accessories and hair follicles increased in
the treated tissues. Collagen fibers increased and have
more organization. In the eucerin and control groups,
situations were same with day 14 with exception hydropic
degeneration (Fig. 4).
On day 28, treatment was seen in about all groups.
DISCUSSION
Zinc oxide ointment is among the most widely used
topical ointments to treat ulcers is that content is 20
percent zinc oxide powder. Protect the surface, being
astringent, antiseptic, and nontoxic relative of the
outstanding characteristics that make the drug as an active
ingredient in health and pharmaceutical compounds
widely used. In this study were used of this ointment as
positive control group too.
Achillea species have been so far reported to contain
diterpenes, sesquiterpenes, flavonoids, lignans,
essential oil and rarely triterpenes (Ahmed et al., 2002;
Barrero et al., 1990; Mockute and Judzentiene, 2003;
Marchart and Kopp, 2003; Aljancic et al., 1996; Maffei
et al., 1994; Oksuz et al., 1991; Kusmenoglu et al., 1995).
For instance, A. vermicularis was shown to have
guaianolide- and germacrenetype sesquiterpenes as well
as flavonoids, whereas A. setacea was reported to contain
sesquiterpenes, essential oils and flavonoids (Marchart
and Kopp, 2003; Oksuz et al., 1991; Unlu et al., 2002). In
addition to extracts, essential oils of the Achillea species
were also analysed. The oil of A. pachycephala was found
to contain 1,8-cineole and camphor as the major
constituents, whereas 1,8-cineole and Artemisia ketone
were major in A. oxyodonta. On the other hand A.
biebersteinii was rich in camphor and borneol followed
by 1,8-cineole. It was stated that all the oils were rich in
oxygenated monoterpenes (Esmaeili et al., 2006). Non-
volatile components of A. biebersteinii afforded in
Curr. Res. J. Biol. Sci., 4(1): 90-95, 2012
94
addition to $-sitosterol, stigmasterol two sesquiterpene
lactones, germacranolide (Badahdah and El-Orfy, 2004).
Essential oil of A. millefolium consists of a number of
monoterpenes such as "-pinene, $-pinene, 1,8-cineole,
camphor and borneol in addition to some sesquiterpene
lactones of germacrene-derivatives (Mockute and
Judzentiene, 2003). Major component in the essential oils
of both A. setacea and A. teretifolia was elucidated to be
1,8-cineole (Unlu et al., 2002) whereas "-pinene, 1,8-
cineole and camphor as well as germacrene D and
bisabolene as the major constituents of ten other Achillea
species (A. biserrata, A. clypeotala, A. crithmifolia, A.
filipendula, A. macrophylla, A. pannonica, A. pyrenaica,
A. sibirica, A. taygetea and A. tenuifolia) (Maffei et al.,
1994). Various biological activity studies were also
completed on Achillea species. The antimicrobial and
antioxidant activities of the essential oil and the
methanolic extract of A. biebersteinii were studied in vitro
by (Baris et al., 2006). The essential oil showed
antimicrobial activity against 8 bacteria sp., 14 fungi sp.
and the C. albicans, whereas the methanolic extract
remained inactive.
When a wound occurs and is exposed to external
environment, it is more prone to attack by microbes,
which invade through the skin and delay the natural
wound healing process. Reactive Oxygen Species (ROS),
are vital part of healing and serve as cellular messengers
that drive numerous aspects of molecular and cell biology.
ROS can trigger the various beneficial pathways of
wound healing, for example, at micromolar concentrations
of hydrogen peroxide can promote Vascular Endothelial
Growth Factor (VEGF) expression in keratinocytes
(Khanna et al., 2001; Prasad and Oberleas, 1974; Nayak
et al., 2009). During the inflammation phase of healing
neutrophils and macrophages are attracted into the injured
tissue by various chemotactic factors. They locate,
identify, phagocytize, kill and digest microorganisms and
eliminate wound debris through their characteristic
“respiratory burst” activity and phagocytosis (Clark and
Moon, 1999). At high concentrations, ROS can induce
severe tissue damage and even lead to neoplastic
transformation, which further impede the healing process
by causing damage to cellular membranes, DNA, proteins
and lipids as well (Martin, 1996). Hence, if a compound
or a plant extract having antioxidant potentials and
antimicrobial activity additionally, it can be a good
therapeutic agent for accelerating the wound-healing
process.
Several preparations containing A. millefolium extract
was quite successfully healed the wounds and scars. The
liniment containing hiperisin oil and A. millefolium extract
patented by Motogna accelerates the healing of wounds
and gives esthetic scars. Since the liniment is applied as
a spray it is easily applied and painless (Motogna, 1971).
The activiy most probably comes from the synergistic
effect of compounds present in the extract and also
additive effect of hiperisin.
According to results reported here yarrow extracts
was found to have better activity on the wound healing
experimental models compared to the other extracts.
ACKNOWLEDGMENT
This study was extracted from research project
which was supported financially by Islamic Azad
university of Tabriz branch so the authors thanks for
mentioned university.
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Ethnopharmacol., 39(1): 31-38.
... The anti-inflammatory effect of A. millefolium EO was exhibited in LPS-stimulated RAW 264.7 macrophages as well (93,94). Besides, it has been shown that a topical cream containing 7.5% alcoholic extract of A. millefolium L. reduced the surface of lower leg ulcers wound by 39% that is significantly more than the control group (95). A. millefolium L. extract, compared to the other Achillea species, was discovered to be more efficient in wound healing of Wistar rats which could be due to the synergistic effects of the active ingredients (96). The extract of A. millefolium L. did not display a cytotoxic effect although it uncovered an enormous potential for enhancing cell migration, cell proliferation, and extracellular matrix production in the treated cells compared to the control group according to the results of an in-vitro study on the effects of hydroalcoholic extract of A. millefolium L. on HSF-PI-16 skin fibroblast cells (97). ...
... When a wound occurs and is exposed to external environment, it is more prone to microbes attack, which invade through the skin and delay the natural wound healing process. Reactive Oxygen Species (ROS), at high concentrations, can induce severe tissue damage and even lead to neoplastic transformation, which further impede the healing process by causing damage to cellular membranes, DNA, proteins and lipids as well [37,38]. Hence, if a compound or a plant extracts has additionally antifungal potentials and antibacterial activity, it can be a good therapeutic agent for the acceleration of the wound-healing process.In conclusion, to our best knowledge, this is the first report of the antimicrobial activity by MIC of different extracts in different solvents such as acetone, ethyl acetate, ethanol, methanol and dimethyl sulfoxide (DMSO) of A. latiloba species. ...
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